SUMMARY Human cytomegalovirus (HCMV) establishes latent infection in hematopoietic progenitor cells. Differentiation of latently infected cells induces reactivation of the HCMV lytic cycle, and subsequent viral spread to multiple organs and tissues. A significant gap in our knowledge is our limited understanding of the mechanisms controlling HCMV reactivation. Understanding the mechanisms by which HCMV reactivates from latency is critical to developing new strategies to limit and control life-threatening disease in immune compromised individuals, such as transplant recipients. A critical step in the establishment of and reactivation from HCMV latency is the expression of the major immediate early proteins (MIE) IE1 and IE2, which are crucial transactivators of the viral lytic cycle. During lytic infection, the major immediate early promoter (MIEP) drives the expression of IE1 and IE2 from a single, alternatively spliced mRNA. During latency, the MIEP is silenced, and IE1 and IE2 are not expressed. HCMV reactivation induces IE1 and IE2 re-expression, therefore it has been presumed that HCMV reactivation requires activation of the MIEP to allow for re-expression of IE1 and IE2. However, our preliminary results show that the MIEP remains silent when latently infected cells are exposed to reactivation stimuli. These data raise the intriguing question: If the MIEP remains inactive, how does HCMV express MIE transcripts during reactivation? Through a series of collaborative studies, we find that alternative MIE promoters we recently identified within the first MIE intron are activated to high levels during reactivation. These transcripts differ from MIEP-derived transcripts solely in their 5? untranslated regions due to alternative transcription start site usage. Importantly, each of the novel transcripts encodes full length IE1 (72-kDa) or IE2 (86-kDa) proteins. Further, our data show that the novel MIE promoters are necessary for IE1 and IE2 re-expression upon reactivation in experimental models of HCMV latency, but not during replication in fibroblasts. Further, we have identified specific host transcription factors (TFs) induced during differentiation that also activate the intronic MIE promoters. We hypothesize that HCMV reactivation from latency requires activation of novel MIE intronic promoters by host TFs associated with myeloid differentiation. In Aim 1, we will define the promoter elements required for IE1 and IE2 re-expression during HCMV reactivation from latency using recombinant viruses in cell lines and primary human hematopoietic progenitors cells. In Aim 2, we will define the role of specific host factors required for differentiation-dependent reactivation of IE1/2 in reactivation. Our proposed studies offer exciting paradigm-shifting insights into the mechanisms that control herpesvirus reactivation. Through collaboration, this multi-PI study is uniquely positioned to make significant advances in our understanding of herpesvirus reactivation and we anticipate this work will broadly inform mechanisms of herpesvirus reactivation important to developing novel strategies to prevent HCMV disease.